Analysis of Nitroxide-Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy.

Measurements of distances in cells by pulsed ESR spectroscopy afford tremendous opportunities to study proteins in native environments that are irreproducible in vitro. However, the in-cell environment is harsh towards the typical nitroxide radicals used in double electron-electron resonance (DEER) experiments. A systematic examination is performed on the loss of the DEER signal, including contributions from nitroxide decay and nitroxide side-chain cleavage. In addition, the possibility of extending the lifetime of the nitroxide radical by use of an oxidizing agent is investigated. Using this oxidizing agent, DEER distance measurements are performed on doubly nitroxide-labeled GB1, the immunoglobulin-binding domain of protein G, at varying incubation times in the cellular environment. It is found that, by comparison of the loss of DEER signal to the loss of the CW spectrum, cleavage of the nitroxide side chain contributes to the loss of DEER signal, which is significantly greater in cells than in cell extracts. Finally, local spin concentrations are monitored at varying incubation times to show the time required for molecular diffusion of a small globular protein within the cellular milieu.

EPR spectroscopy shows that the blood carrier protein, human serum albumin, closely interacts with the N-terminal domain of the copper transporter, Ctr1.

Copper is an essential metal whose localization within the cells must be carefully controlled to avoid copper dependent redox cycling. Although most of the key proteins involved in cellular copper transfer have been identified, fundamental questions regarding the copper transfer mechanism have yet to be resolved. One of the blood carrier proteins believed to be involved in copper transfer to the cell is human serum albumin (HSA). However, direct evidence for close interaction between HSA and the extracellular domain of the copper transporter Ctr1 has not yet been found. By utilizing EPR spectroscopy, we show here that HSA closely interacts with the first 14 amino acids of the Ctr1, even without the presence of copper ions.